We continue to identify and characterize oncogenic events and their consequences in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Progress during the past year includes the following: Activation of NF-kappaB (NFkB) pathway by mutations in MM tumors and cell lines. We initially found that two human MM cell lines (HMCL) have constitutively activated the NFkB pathway by over-expression of NFkB-inducing kinase (NIK) due to either a translocation or gene amplification. An inhibitor of IkB kinase beta (IKK2) caused cessation of cell division or death in approximately 50% of HMCL, including the two HMCL that over-express NIK. The effect of this inhibitor on several HMCL was used to generate an 11 gene molecular signature of NFkB activity. Using this NFkB index to analyze data from gene expression arrays, we found that 50% of HMCL, 82% of MM tumors, 95% of MGUS tumors, and all normal bone marrow plasma cells have substantial activation of the NFkB pathway. Focusing on NIK and other genes that regulate the NFkB pathway for mutations, we found that the expression of two positive NFkB regulators NIK and CD40 is elevated in some MM or HMCL samples, while four negative regulators TRAF3, CYLD, BIRC2, and BIRC3 are absent or inactivated in other samples. In addition, over-expression of the NFKB1 gene or truncated forms of the NFKB2 gene were found to activate the NFkB pathway in some HMCL. In all cases, these mutations were found in tumors or HMCL that have a high NFkB index. The prevalence of mutations is estimated to be 15-20% in untreated MM tumors, and >35% in HMCL, suggesting that the mutations are likely to occur during tumor progression. Most mutations in HMCL activate both the classical and alternative NFkB pathways, but our data suggest that the classical pathway activation may be most critical in view of results with the IKK2 inhibitor that selectively targets the classical pathway. Others had reported that activation of the NFkB pathway by extrinsic signals from normal bone marrow stromal cells is critical for survival of normal mouse plasma cells. Therefore, our results suggest that activation of the NFkB pathway - either by extrinsic signals or intrinsic mutations that presumably decrease the dependence of the tumor on the bone marrow microenvironment is important for the survival and growth of most MGUS and MM tumors. The possible addiction of MGUS and MM tumors to an activated NFkB pathway suggests that these tumors may be susceptible to a variety of NFkB pathway inhibitors that are being developed by pharmaceutical companies. Dysregulation of the RB pathway in HMCL and proliferative MM tumors. The dysregulation/over-expression of a CYCLIN D gene is a unifying event in virtually all MGUS and MM tumors. In addition, we have found that p16INK4A expression is absent or extremely low in most HMCL and MM tumors, even though the p16 gene is methylated in >90% of HMCL but only 20-30% of MGUS and MM tumors; this indicates that the lack of p16 expression does not require methylation. Despite the dysregulation of a CYCLIN D gene and low or absent expression of p16, most MM tumors have an extremely low proliferation index. We have determined that the expression p18INK4C, which seems to be a key regulator of the G1>S transition in normal plasma cells, provides an important marker of proliferation. In approximately 30% of HMCL and nearly 10% of proliferative MM tumors, the bi-allelic deletion of p18 appears to be a critical event that is associated with increased proliferation. Expression of exogenous p18 in HMCL that express little or no p18 markedly decreases proliferation in HMCL due to a block in the G1>S transition. Paradoxically, however, about 60% of both HMCL and MM tumors that are hightly proliferative have substantially increased levels of p18 compared to normal plasma cells, suggesting that these tumor cells are insensitive to the increased levels of p18 that occur as a consequence of increased proliferation. In fact increased expression of p18 RNA is associated with decreased patient survival. About 15% of HMCL and proliferative tumors that express high levels of p18 have inactivated RB1, which would explain their insensitivity to high p18 levels. Therefore, we conclude that an increase in proliferation with progression of MM tumors is explained in at least some cases by sequential steps (inactivation of p18 or RB1) that dysregulate the RB pathway. We are continuing efforts to identify additional mechanisms responsible for increased proliferation when there are high p18 levels